Microvesicle Formation Research Articles

Induction of ectosome formation by binding of phospholipases D from Loxosceles venoms to endothelial cell surface: Mechanism of interaction

Members of the phospholipase D (PLD) superfamily found in Loxosceles spider venoms are potent toxins with inflammatory and necrotizing activities. They degrade phospholipids in cell membranes, generating bioactive molecules that activate skin cells. These skin cells, in turn, activate leukocytes involved in dermonecrosis, characterized by aseptic coagulative necrosis. Although the literature has advanced in understanding the structure-function relationship, the cell biology resulting from the interactions of these molecules with cells remains poorly understood. In this study, we show that different cells exposed to recombinant PLDs bind these molecules to their plasma membrane, leading to the subsequent organization of extracellular microvesicles/ectosomes. The binding occurs as quickly as five minutes or less after exposure, increases over time, and eventually, the PLDs are expelled from the cell surface without generating cytotoxicity. PLDs are not endocytosed, nor do they spatially colocalize with acidic organelles in the intracellular environment. At least two regions of PLDs – the domain involved in magnesium ion coordination and the choline binding site – appear to play a role in cell surface binding and ectosome organization. However, the amino acids involved in catalysis do not participate in these events. The binding of these PLDs to the cell membrane, independent of catalytic activity, is sufficient to trigger intracellular signaling and enhance the expression of the pro-inflammatory IL-8 gene. These results are supported by the observation that isoforms of PLDs lacking catalytic activity induce an inflammatory response in vivo when injected into the skin of rabbits, without causing dermonecrosis. Our data indicate that these PLDs bind to the surface of target cells, promoting the organization of extracellular vesicles/ectosomes. Subsequently, these events activate pro-inflammatory genes and induce an inflammatory response in vivo. The binding to cells is not dependent on amino acids involved in catalysis but rather on amino acids involved in magnesium coordination. The binding of PLDs to the cell surface, formation of ectosomes, and activation of cells appear to initiate signals involved in inflammatory responses that can lead to dermonecrosis in accidents. This correlation is supported by experimental observations indicating that the events of toxin binding to cells, formation of microvesicles, and inflammatory responses observed both in vitro and in vivo are interconnected.

Autographa californica multiple nucleopolyhedrovirus ac106 is required for the nuclear egress of nucleocapsids and intranuclear microvesicle formation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf106 (ac106) is highly conserved in baculoviruses. Previous studies have shown that ac106 is required for the production of infectious budded virions (BVs). However, the functional role of ac106 in virion morphogenesis remains unknown. In this report, an ac106 knockout virus and an ac106 repair virus were constructed. The effect of ac106 deletion on virion morphogenesis was investigated, and the expression and subcellular localization of the Ac106 protein were characterized. Our data indicated that ac106 is required for the nuclear egress of nucleocapsids and intranuclear microvesicle formation, as well as subsequent BV and occlusion-derived virion (ODV) production and the embedding of ODVs into polyhedra. Ac106 is a baculovirus late protein that is concentrated in discrete foci of virus-induced membrane structures in the intranuclear ring zone of virus-infected cells. Further studies on the relationship between Ac106 and four other proteins that are also required for intranuclear microvesicle formation, Ac75, Ac76, Ac93, and P48 (Ac103), revealed that Ac106 is associated with Ac75, Ac76, Ac93, P48, and itself. Ac106 is required for Ac75, Ac93, and P48 accumulation in foci of virus-induced intranuclear membrane structures and the intranuclear transport of Ac76. Analysis of the subcellular localization of ODV integral envelope proteins upon deletion of the genes required for intranuclear microvesicle formation indicated that intranuclear microvesicle formation may be essential for ODV integral envelope protein transport into the nucleus, supporting the hypothesis that intranuclear microvesicles originate from the nuclear membrane.IMPORTANCEBaculovirus occlusion-derived virions (ODVs) are known to acquire their envelopes from virus-induced intranuclear microvesicles within the nucleoplasm, and this strategy of intranuclear envelopment of nucleocapsids to form virions is unique among viruses. However, the mechanism of ODV morphogenesis, particularly intranuclear microvesicle formation, remains unclear. In this study, we identified ac106 as the fifth gene, in addition to ac75, ac76, ac93, and p48 (ac103), which are required for intranuclear microvesicle formation. Further studies on the relationship between ac106 and the other four genes, as well as the effect of ac106 or ac75 deletion on the localization of ODV integral envelope proteins, indicated that intranuclear microvesicle formation may be essential for the transport of ODV integral envelope proteins into the nucleus, which strongly supports the hypothesis that intranuclear microvesicles originate from the nuclear membrane. These findings greatly enhance our understanding of the molecular mechanism of baculovirus ODV morphogenesis.

Phosphatidylserine-binding molecules induce the formation of tumor cell-derived microvesicles for enhanced cancer immunotherapy

Engineered microvesicles (MVs) derived from tumor cells, which preserve tumor-specific characteristics, have been extensively investigated as delivery systems or vaccines for cancer therapy. However, the negatively charged phosphatidylserine (PS) exposed on the surface of tumor-derived MVs can induce immune tolerance, evade immune responses and dampen immunotherapy effectiveness. Here, we report a functional molecule (FaZn) that not only actively targets PS exposed on tumor cell surfaces, inducing membrane blebbing and producing PS-blocked tumor cell-derived MVs (FaZn-MVs) but also directly generates stable PS-blocked MVs in situ within tumors, serving as an antigen repository. FaZn-MVs not only activate T-cell-mediated immune responses akin to a prophylactic vaccine, demonstrating robust protection against tumors but also suppress tumor growth in mice and offer long-term immune surveillance. Furthermore, when FaZn-MVs are combined with programmed death ligand 1 (PD-L1) inhibitors, they enhance potent antitumor immune responses, leading to significant improvements in survival and immune memory. This work establishes a promising method for the in vivo generation of personalized tumor microvesicle vaccines with PS blockade, revealing a promising strategy for activating antitumor immune responses.

Evaluation of circulating microvesicles and their procoagulant activity in patients with COVID-19

ObjectiveSeveral pathological conditions trigger the formation of microvesicles (MVs), including infectious diseases such as COVID-19. The shedding of MVs increases the levels of inflammatory factors (e.g., interleukin-6; IL-6) and ultimately leads to an inflammatory cascade response, while also increasing the procoagulant response. The current study aimed to evaluate the level of circulating MVs and their procoagulant activity as well as the serum level of IL-6 in patients with COVID-19 and healthy controls. In this case-control study, 65 patients with COVID-19 and 30 healthy individuals were sampled after obtaining written informed consent. MVs counting was measured using conjugated CD61, CD45, CD235a, and Annexin-V antibodies. Additionally, the procoagulant activity of MVs and the IL-6 level were estimated using enzyme-linked immunosorbent assay (ELISA).ResultsThe majority of MVs were platelet-derived MVs (PMVs). Patients with COVID-19 had significantly higher levels of MVs, procoagulant MVs, and IL-6 compared to healthy controls (p < 0.001). MVs were significantly correlated with procoagulant MVs, D-Dimer levels, fibrinogen, and IL-6, but not with platelet, lymphocyte, and neutrophil counts.ConclusionElevated levels of procoagulant MVs and their association with inflammatory and coagulation markers in patients with COVID-19 are suggested as a novel circulatory biomarker to evaluate and predict the procoagulant activity and severity of COVID-19.

A CRM1-dependent nuclear export signal in Autographa californica multiple nucleopolyhedrovirus Ac93 is important for the formation of intranuclear microvesicles.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.

The ras-related protein RAB22A interacts with hypoxia-inducible factor 1-alpha (HIF-1α) in MDA-MB-231 breast cancer cells in hypoxia.

Recent studies suggest that hypoxia-inducible factor 1-alpha (HIF-1α) and the small GTPase protein Ras-related protein Rab-22A (RAB22A) may be colocalized in the cytoplasm and that as a conequence they may enhance the formation of microvesicles in breast cancer cells under hypoxia. Therefore, we sought to determine whether these two proteins are present in intracellular complexes in breast carcinoma cells. Evaluation using molecular docking indicated that HIF-1α and RAB22A interact with each other. Co-immunoprecipitation of endogenous or ectopically expressed HIF-1α and RAB22A proteins in MDA-MB-231 breast cancer cells or HEK-293T cells demonstrated that endogenous HIF-1α and RAB22A can form an intracellular complex; however, transiently expressed HIF-1α and RAB22A failed to interact. Investigating RAB22A and HIF-1α interactions in various cancer cell lines under hypoxia may shed light on their roles in cancer cell survival and progression through regulation of intracellular trafficking by HIF-1α under hypoxic conditions. Our study is the first to reveal the potential involvement of HIF-1α in intracellular trafficking through physical interactions with the small GTPase protein RAB22A. We discuss the implications of our work on the role of exosomes and microvesicles in tumor invasiveness.

Platelets: Physiology and Biochemistry.

This article represents a republication of an article originally published in STH in 2005. This republication is to help celebrate 50 years of publishing for STH. The original abstract follows.Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross-talk to other blood and vascular cells. Stimulated "sticky" platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

#4049 NEFECON® TREATMENT LIKELY MODULATES DOWNSTREAM PATHWAYS OF KIDNEY INFLAMMATION AND FIBROSIS IN IGA NEPHROPATHY

Abstract Background and Aims The lifetime risk of kidney failure in patients with IgA nephropathy (IgAN) is substantial. Since the original description of the disease, over 50 years ago, there has been little progress in how we manage this important cause of kidney failure in young adults. KDIGO 2021 Clinical Practice Guidelines recommend goal-directed supportive care as the treatment with the strongest evidence base for the management of all patients with IgAN. The NEFIGAN trial (NCT01738035) tested the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver budesonide to the gut-associated lymphoid tissue-rich distal ileum in patients with IgAN in addition to optimised supportive care. The trial comprised a 6-month run-in, 9-month treatment, and a 3-month follow-up phase. Forty-eight patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day, and 50 patients received placebo. The headline result of the study was that Nefecon® 16 mg/day, added to optimised RAS blockade, reduced proteinuria and stabilised eGFR in patients with IgAN. These findings have now been replicated in the NefIgArd study, which reported in 2021 and provided the basis for the recent FDA and EMA approval of Nefecon® as a treatment for patients with IgAN at high risk of progressive disease. In this study, we determined the composition of urinary proteins from patients treated with placebo and 16 mg of Nefecon® in the NEFIGAN trial using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Method Urine samples from 18 patients from each of the placebo and 16-mg arms of the NEFIGAN trial collected at the start of treatment (SOT) and end of treatment (EOT) were analysed. Patients were only included if they had received at least 8 months of treatment and their urine sample was taken up to 2 days after the completion of tapering. Urine samples were treated to remove abundant albumin and immunoglobulins. The remaining proteins were desalted and then precipitated with 5% trichloroacetic acid and washed twice with ice-cold acetone. The air-dried pellets were dissolved in 45 µL of 100 mM triethylammonium bicarbonate buffer, reduced with 2 mM TCEP (tris(2-carboxyethyl)phosphine), and alkylated with 15 mM chloroacetamide. 500 ng of Sequencing Grade Trypsin was added for enzymatic digestion, carried out overnight at 37°C. The samples were dried to completeness and re-solubilised in 10 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid). LC-MS/MS analysis was performed on an Q Exactive mass spectrometer equipped with a Digital PicoView source and coupled to a nanoAcquity UPLC. For protein identification and quantification, raw data were processed with FragPipe (V16) having at least two peptides per protein. Protein intensities were reported in the combined_protein.txt file generated by the FragPipe: These were Log_2-transformed and internally normalised against a group of peptides found in all samples, The normalised protein abundance at SOT was subtracted from the abundance at EOT for each patient, A probabilistic dropout model was fitted to the data to estimate fold changes between the treatment and placebo groups at EOT, The proteins were ranked using the t-statistic and a gene-set enrichment analysis was performed to determine the gene sets significantly affected by the treatment compared with the placebo group. Results Gene-ontology analysis revealed that treatment with 16 mg of Nefecon® led to a significant enrichment of multiple pathways (n = 57) involved in several processes previously shown to be important in the pathogenesis of kidney injury in IgAN. These included epigenetic pathways (n = 13), microvesicle formation (n = 3), kidney remodelling (n = 9), and regulation of local immune and inflammatory responses (n = 32). Conclusion We have previously shown that 16 mg of Nefecon® for 9 months positively impacts on multiple biomarkers of IgAN severity. These urine proteomic data support the positive impact of Nefecon® on downstream pro-inflammatory and profibrotic pathways within the kidneys. These data will be validated in the biomarker analyses currently underway as part of the NefIgArd study.

Tyrame [N-(3-hydroxy-1:3:5(10)-estratrien-17β-yl)-4-hydroxy-phenethylamine], antithrombotic aminoestrogen that decreases microvesicle formation.

Estrogens that are used as contraceptives or in replacement therapy are associated with an increase in the risk for developing thrombosis, mainly during the first year of treatment and in women with associated risk factors. To synthesize, characterize and identify the anticoagulant, antiplatelet aggregation and microvesicle-reducing effect of the new aminoestrogen Tyrame. CD1 strain mice were used, which had Tyrame (0, 1 and 2 mg/100 g) subcutaneously administered. At 24 h, a blood sample was obtained to determine whole-blood clotting time, microvesicles concentration and inhibitory effect on platelet aggregation. Blood clotting time increased up to 1.5 times in comparison with the control. Platelet aggregation inhibition had different magnitude depending on the agonist agent employed, and was complete with collagen. Both effects had a dose-dependent relationship. The microvesicles decreased up to six times with respect to the control. Tyrame reduces platelet aggregation and microvesicle formation, which emphasizes its potential therapeutic utility as an estrogen free of thrombotic effects.

The role of tumor-platelet interplay and micro tumor thrombi during hematogenous tumor metastasis.

In addition to their pivotal roles in coagulation and thrombosis, platelets are crucial in tumor progression, with plenty of clinical and experimental data demonstrating that the interplay of platelets and tumor cells is essential for hematogenous tumor metastasis. After detach from primary sites, tumor cells intravasate into the blood circulation becoming circulating tumor cells and induce platelet activation, aggregation and encasement around tumor cells to form micro tumor thrombi, which create a permissive tumor microenvironment for metastasis. Platelets in micro tumor thrombi protect tumor cells from immune surveillance and anoikis (detachment-triggered apoptosis) through various pathways, which are significant for tumor cell survival in the bloodstream. Moreover, platelets can facilitate tumor metastasis by expediting epithelial-mesenchymal transition (EMT), adhesion to the endothelium, angiogenesis, tumor proliferation processes and platelet-derived microvesicle (PMV) formation. Here, we provide a synopsis of the current understanding of the formation of micro tumor thrombi and the role of micro tumor thrombi in tumor hematogenous metastasis based on the tumor-platelet interplay. We also highlight potential therapeutic strategies targeting platelets for tumor treatment, including cancer-associated platelet-targeted nanomedicines.

Assessment of microvesicles from different cell origins in patients with psoriasis: evidence of thrombogenic, proinflammatory microenvironment in the absence of established cardiovascular disease.

Psoriasis is associated with increased cardiovascular risk. Endothelial, platelet, and erythrocyte microvesicles (MVs) are novel biomarkers of endothelial dysfunction and thromboinflammation. We explored whether MVs of different cell types are elevated in patients with psoriasis, and investigated potential associations with disease severity and macrovascular function. Endothelial, platelet and erythrocyte MVs were measured using a standardized flow cytometry protocol in psoriasis patients and controls free from established cardiovascular disease. Carotid intima-media thickness (IMT) and pulse wave velocity (PWV) were measured as markers of subclinical atherosclerosis and arterial stiffness. Psoriasis severity was assessed with PASI (Psoriasis Area Severity Index). Both platelet (p < 0.001) and erythrocyte MVs (p = 0.046), yet not endothelial MVs, were significantly increased in patients with psoriasis (n = 41) compared with controls (n = 41). Patients with higher PASI (≥10) presented significantly higher levels of ErMVs compared to those with lower PASI (<10) (p = 0.047). Carotid IMT and PWV were comparable between psoriasis patients and controls and did not significantly correlate with MVs. In the multivariate analysis, psoriasis was identified as an independent predictor of both platelet (p < 0.001) and erythrocyte MVs (p = 0.043), while hypertension was independently associated with endothelial MVs (p < 0.001). Increased formation of platelet and erythrocyte MVs may be evident in psoriasis patients and is indicative of prothrombotic, proinflammatory microenvironment, even in the absence of subclinical macrovascular dysfunction and before the clinical onset of overt cardiovascular complications. Potential mechanistic links and prognostic implications of increased MVs in psoriasis warrant further investigation.

Engineering caveolin-mediated endocytosis in Saccharomyces cerevisiae

As a potential substitute for fatty acids, common low-cost oils could be used to produce acetyl-CoA derivatives, which meet the needs of low-cost industrial production. However, oils are hydrophobic macromolecules and cannot be directly transported into cells. In this study, caveolin was expressed in Saccharomyces cerevisiae to absorb exogenous oils. The expression of caveolin fused with green fluorescent protein showed that caveolin mediated the formation of microvesicles in S. cerevisiae and the addition of 5,6-carboxyfluorescein showed that caveolae had the ability to transport exogenous substances into cells. The intracellular and extracellular triacylglycerol levels were then detected after the addition of soybean oil pre-stained with Nile Red, which proved that caveolae had the ability to absorb the exogenous oils. Lastly, caveolin for oils absorption and lipase from Bacillus pumilus for oil hydrolysis were co-expressed in the naringenin-producing Saccharomyces cerevisiae strain, resulting in naringenin production increasing from 222 mg/g DCW (dry cell weight) (231 mg/L) to 269 mg/g DCW (241 mg/L). These results suggested that the caveolin-mediated transporter independent oil transport system would provide a promising strategy for the transport of hydrophobic substrates.

Natural killer cells: origin, phenotype, function

Natural killer cells (NK) are innate immune lymphocytes produced in the bone marrow. Isolation of NK cells as a separate population of lymphocytes is related to discovery of their ability to induce the death of tumor cells without prior sensitization. In this review, an attempt was made to systematize the numerous data on the biology of NK cells presented in the literature. The authors consider the stages of NK cells` differentiation from a common lymphoid progenitor (CLP) in the bone marrow, describe two functionally different populations of mature NK cells – CD56brightCDl6- and CD56dimCD16+. In addition, the role of cytokines and chemokines in the development of NK cells is discussed. The review includes data on the spectrum of molecules expressed by NK cells: adhesion molecules (LFA-1, LFA-2, LFA-3; αMβ2, αXβ2, L-selectin, VLA-4, VLA-5; PECAM-1; CEACAM-1), cytokine receptors (IL-1R, IL-2ra, IL-2Rb/IL-2Rc, IL-6Rα, IL-7Ra, IL-8R, IL-10R, IL-12Rβ1, IL-15ra, IL-18R, IL-21ra, IFNGR2, TGFBR, c-Kit, CXCR1, CXCR3, CXCR4, CCR4, CCR5, CCR6, CCR7, IChemR23, CX3CR1), as well as receptors that regulate the activity of NK cells (LILRB1, LILRB2, LILRB4; KIR2DL1-5; KIR2DS1-5; KIR3DL1-3; KIR3DS1; NKG2A, NKG2C, NKG2D; Siglec7, Siglec9; CD16; NKRP-1; TIGIT; TACTILE; NKp30, NKp44, NKp46, NKp80; LAIR-1; PD-1; TIM-3; 2B4; TLR1-9). The authors also examine the mechanisms of implementing cytotoxic activity by NK cells, including cytotoxicity, via expression of MHC-I-specific receptors, CD16 Fc receptors, receptors and ligands of apoptosis (Fas-FasL and TRAIL-TRAILR) as well as other receptors. The review describes in detail the structure of immunological synapse between the NK cell and target cell, receptor interactions, and the role of the cytoskeleton in its formation. The data are summarized on the variants of exocytosis of lytic granules by NK cells, including complete or partial fusion of vesicles with the plasma membrane, exocytosis of vesicles containing perforin and FasL, and the formation of microvesicles containing granzyme B. The review also describes data on ability of NK cells to maintain activated state for a long time, as well as to maintain contact with several targets at the same time. In addition to the functions inherent in natural killers as cells of innate immunity, the authors point out their ability to exhibit the features of cells of adaptive immunity. In general, a variety of mechanisms that regulate the activity of NK cells may complement the specific functions of lymphocytes, thus making the immune system more efficient.

Neutrophil microvesicles and their role in disease.

Microvesicles are formed through shedding from the plasma membrane, a process shared by almost all human cells. Microvesicles are highly abundant and have been detected in blood, urine, cerebrospinal fluid, and saliva. They contain a library of cargo derived from their parental cell during formation, including proteases, micro-RNAs and lipids and delivery of this parental cell-derived cargo to other cells can alter target cell function and drive disease. Cell specific molecules on the surface of microvesicles, obtained during microvesicle formation, allows their parental cell to be identified and populations of microvesicles to be investigated for roles in the pathogenesis of various diseases. For instance, recent work by our group has identified a role for neutrophil microvesicles in atherosclerosis. Microvesicle profiles could in future be associated with certain diseases and act as a biomarker to allow for earlier diagnosis. This short review will discuss some of the processes central to all microvesicles before focusing on neutrophil microvesicles, their potential role in cardiovascular disease and the mechanisms that may underpin this.

The amino acids of Autographa californica multiple nucleopolyhedrovirus P48 critical for the association with Ac93 are important for the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) undergoes a biphasic life cycle with the production of two physically and functionally distinct virions: budded virions (BVs) and occlusion-derived virions (ODVs). Nuclear egress of nucleocapsids and intranuclear microvesicle formation are critical for the morphogenesis of BVs and ODVs, respectively, but the mechanisms and details of these two processes remain unknown. Our previous studies have shown that AcMNPV p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and protein P48 associates with Ac93, which is also involved in the above processes in virion morphogenesis. In this study, we present evidence that alanine substitution for residues N318, V319, C320, R321, and I323 of P48 disrupted the association with Ac93. Moreover, mutation of these residues blocked the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. These results suggested that the association between P48 and Ac93 may be important for both BV and ODV morphogenesis.

Evidence for Systemic Reactive Oxygen Species in UVB-mediated Microvesicle Formation.

Recent studies have implicated subcellular microvesicle particles (MVP) in the ability of ultraviolet B radiation to exert both local and systemic effects. Indeed, UVB generates MVP (UVB-MVP) in human skin and systemically following phototherapy. The current studies were designed to test the hypothesis that the ability of UVB to generate MVP was dependent upon reactive oxygen species (ROS). To that end, we tested urine samples from subjects undergoing UVB phototherapy for the presence of isoprostanes as well as the oxidized guanosine derivative 8OHdG. We also conducted a clinical study in which volar forearms of subjects were treated with localized UVB and erythema/MVP measured. The same cohort was then treated with 7 days of vitamin C (2 g day-1 ) and vitamin E (1000 IU day-1 ), and UVB-induced MVPs tested on the contralateral forearm. Urine specimens from subjects undergoing phototherapy were found to have increased levels of isoprostanes and 8OHdG, with maximal levels noted 8-16 h post-treatment. Treatment with antioxidant vitamins resulted in diminished UVB-generated skin MVP to baseline levels. These studies suggest that whole-body UVB generates a systemic pro-oxidative response, and that antioxidants can attenuate localized skin UVB-MVPs.

Features of apoptosis and blebbing of the lymphocyte plasma membrane in bronchial asthma

Given a persistent global trend towards an increase in the number of patients with bronchial asthma (BA) over the past decades, researchers are facing challenges related to a comprehensive study of the pathogenesis of BA. Numerous studies have shown that BA is associated with long-term persistence of leukocytes (lymphocytes, macrophages, and eosinophils) in the bronchial tissues. However, the causes of this phenomenon remain understudied. The article provides an overview of modern research on the mechanisms of disorders of lymphocyte apoptosis in patients with BA. Our study considers the main mechanisms of molecular regulation of lymphocyte apoptosis, including transcription factors, the Fas/FasL system, and bcl-2/bcl-XL factors. We present the data on the role of reduced lymphocyte apoptosis in the formation of a severe BA phenotype. Taking into account high prevalence of obesity among patients with BA, we analyzed a few existing articles on the apoptosis of immunocompetent cells in obesity. In addition, the article highlights the key mechanisms of development of lymphocyte plasma membrane blebbing (PMB) with formation of microvesicles, as well as their influence on the course of pathological processes in BA. The authors believe that further in-depth study of apoptosis, lymphocyte necrosis, and plasma membrane blebbing can help improve the principles of diagnosis and treatment of BA.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Vesiculation red blood cells. Its role in donor erythrocytes components

The formation of microvesicles by blood cells: monocytes, platelets, granulocytes, erythrocytes and endothelial cells is the most important feature of intercellular interactions. Red blood cells form microvesicles to remove damaged cell components, such as oxidized hemoglobin and damaged membrane components, and thus extend their functioning. Two hypotheses have been put forward for the formation of microvesicles: programmed cell death (eryptosis) and clustering of the band 3 protein as a result of disruption of intercellular interactions. In the process of eryptosis, damage to hemoglobin and a change in the pathways of phosphorylation of membrane proteins, primarily protein of strip 3, weaken the strong bonds between the lipid bilayer and the cytoskeleton, which is accompanied by the transformation of the membrane, the formation of protrusions and their transformation into microvesicles. It was found that the formation of microvesicles by red blood cells is impaired in patients suffering from various pathologies of red blood cells: sickle cell anemia, glucose-6-dehydrogenase deficiency, spherocytosis, and malaria. Studies of the last decade show that a violation of the interaction between the membrane and the cytoskeleton is probably the main mechanism, since it is confirmed by data obtained in the study of structural changes in red blood cells of donor hemocomponents stored in a blood bank. Currently, studies on the effect of microvesicles on the safety of erythrocyte-containing blood components have become widespread. A discussion was resumed on the relationship between the number of accumulated microvesicles in blood components and the effectiveness of donor components for patients during transfusion, depending on the shelf life of the components. Detailed data on proteomic, lipidomic and immunogenic comparisons of microvesicles obtained from various sources are convincing in the identification of trigger stimuli causing the generation of microvesicles. Elucidation of the contribution of microvesicles obtained from red blood cells to inflammation, thrombosis, and autoimmune reactions confirms the need to further study the mechanisms and consequences of the generation of microvesicles by red blood cells of donor components used for transfusion medicine.